Insulin resistance is influenced by genetic and environmental inputs (GxE). Critically ‘environment’ also includes an individual's gut microbiome which has emerged as a critical driver of host metabolic health, with some taxa being beneficial and others detrimental. Crucially these studies largely neglect the impact of host genetics on the microbiome. To study the GxE of complex diseases, our laboratory utilises a population of diversity outbred mice (Diversity Outbred Australia; DOz). We performed genetic mapping of insulin sensitivity in 670 DOz mice and identified a significant quantitative trait locus (QTL) within the α-defensin locus. Alpha-defensins are an ancient group of antibacterial peptides which modulate the microbiome. Single nucleotide polymorphism (SNP) analysis of DOz mice identified rs238754102 as insulin sensitising, and 16s amplicon sequencing of mice carrying this variant revealed an enrichment for metabolically protective microbes including Akkermansia muciniphila. Furthermore, cage-mates of these mice also exhibited enhanced insulin sensitivity and increased A. muciniphila abundance suggesting a microbiome mediated phenotype. To further test the association between defensins and insulin sensitivity, and identify metabolically important isoforms we performed intestinal tissue proteomics in 7 DOz founder strains. This identified α-defensin-26 as positively contributing to whole-body insulin sensitivity. Importantly our insulin sensitivity QTL also correlated with α-defensin-26 expression, and the abundance of various tight junction proteins suggesting increased gut-barrier integrity. Based on our data, and to test causality, we then synthesised α-defensin-26 via native chemical ligation techniques and investigated the protective potential of exogenous α-defensin-26 against diet-induced obesity and insulin resistance in two strains with naturally varied α-defensin-26 protein expression. Diet supplementation with α-defensin-26 modulated the microbiome, metabolic health and plasma bile acids in a strain-dependent manner suggestive of precision medicine. Our data bring together genetic mapping, multiomics, and peptide chemistry to suggest a metabolically beneficial post-biotic.